Recent reports have challenged the notion that retroviruses and retroviral vectors integrate randomly into the host genome. These reports pointed to a strong bias toward integration in and near gene coding regions and, for gammaretroviral vectors, around transcription start sites. Here, we report the results obtained from a large-scale mapping of 572 retroviral integration sites (RISs) isolated from cells of 9 patients with X-linked SCID (SCID-X1) treated with a retrovirus-based gene therapy protocol. Our data showed that two-thirds of insertions occurred in or very near to genes, of which more than half were highly expressed in CD34 + progenitor cells. Strikingly, one-fourth of all integrations were clustered as common integration sites (CISs). The highly significant incidence of CISs in circulating T cells and the nature of their locations indicate that insertion in many gene loci has an influence on cell engraftment, survival, and proliferation. Beyond the observed cases of insertional mutagenesis in 3 patients, these data help to elucidate the relationship between vector insertion and long-term in vivo selection of transduced cells in human patients with SCID-X1.

Integration-site selection by retroviruses and retroviral vectors has gained increased scientific interest. Foamy viruses (FVs) constitute a unique subfamily (Spumavirinae) of the family Retroviridae, for which the integration pattern into the human genome has not yet been determined. To accomplish this, 293 cells were transduced with FV vectors and the integration sites into the cellular genome were determined by a high-throughput method based on inverse PCR. For comparison, a limited number of murine leukemia virus (MLV) and human immunodeficiency virus (HIV) integration sites were analysed in parallel. Altogether, 628 FV, 87 HIV and 141 MLV distinct integration sites were mapped to the human genome. The sequences were analysed for RefSeq genes, promoter regions, CpG islands and insertions into cellular oncogenes. Compared with the integration-site preferences of HIV, which strongly favours active genes, and MLV, which favours integration near transcription-start regions, our results indicate that FV integration has neither of these preferences. However, once integration has occurred into a transcribed region of the genome, FVs tend to target promoter-close regions, albeit with less preference than MLV. Furthermore, our study revealed a palindromic consensus sequence for integration, which was centred on the virus-specific, four-base-duplicated target site. In summary, it is shown that the integration pattern of FVs appears to be unique compared with those of other retroviral genera. INTRODUCTIONThere are two main reasons for interest in the genetic mapping of retroviral integration sites into the human genome. With respect to applied research, the adverse effects that occurred after gene correction of X-linked severe combined immunodeficiency by gammaretroviral murine leukemia virus (MLV)-derived vectors has stimulated research into alternative retroviral vector systems that show a more inert integration profile (Hacein-Bey-Abina et al., 2003). The MLV vector was used to introduce the corrected gene into haematopoietic stem cells of boys with common c-chain deficiency of the interleukin receptor (Hacein-Bey-Abina et al., 2002). In a minority of patients, a lymphoproliferative disease developed that was explained in part by the integration of the vector in the vicinity of cellular proto-oncogenes, including the LMO-2 proto-oncogene (Hacein-Bey-Abina et al., 2003). This is believed to have stimulated the proto-oncogene promoter by enhancer function of the vector virus U3 elements in the long terminal repeat (LTR) von Kalle et al., 2004). Preferential integration of MLV into the promoter regions of actively transcribed genes is thought to be responsible, at least in part, for this gene activation (Wu et al., 2003).Besides this applied aspect, there is an interest in understanding the retroviral integration pattern from a more basic scientific point of view. Investigating retroviral integration was, until recently, limited to in vitro assays using recombinant enzymes and oligonucleotides or in vivo assays usin...

Tandem dimer Tomato (tdTomato) provides a useful alternative to enhanced green fluorescent protein (eGFP) for performing simultaneous detection of fluorescent protein in histological sections together with fluorescence immunohistochemistry (IHC). eGFP has many properties that make it useful for cell labeling; however, during simultaneous fluorescence IHC, the usefulness of eGFP may be limited. This limitation results from a fixation step required to identify eGFP in histological tissue sections that can mask antibody epitopes and adversely affect staining intensity. An alternative fluorescent protein, tdTomato, may assist concurrent detection of fluorescent protein within tissue sections and fluorescence IHC, because detection of tdTomato does not require tissue fixation. Tissue sections were obtained from various organs of mice ubiquitously expressing eGFP or tdTomato that were either unfixed or fixed with 4% paraformaldehyde. These tissues later were combined with fluorescence IHC. Both eGFP and tdTomato displayed robust signals in fixed frozen sections. Only tdTomato fluorescence, however, was detected in unfixed frozen sections. Simultaneous detection of fluorescence IHC and fluorescent protein in histological sections was observed only in unfixed frozen tdTomato tissue. For this reason, tdTomato is a useful substitute for eGFP for cell labeling when simultaneous fluorescence IHC is required.

The transcriptional targeting of HSV lytic infection to MDK-expressing tumor cells is feasible. oHSV-MDK-34.5 shows enhanced anti-tumor effects both in vitro and in vivo. Further studies are warranted and may lead to its use in clinical trials.

Bacterial methionine aminopeptidase (MAP) is a protease that removes methionine from the N termini of newly synthesized bacterial proteins after the peptide deformylase enzyme cleaves the formyl group from the initiator formylmethionine. MAP is an essential bacterial gene product and thus represents a potential target for therapeutic intervention. A fundamental challenge in the antibacterial drug discovery field is demonstrating conclusively that compounds with in vitro enzyme inhibition activity produce the desired antibacterial effect by interfering with the same target in whole bacterial cells. One way to address the activity of inhibitor compounds is by profiling cellular biomarkers in whole bacterial cells using compounds that are known inhibitors of a particular target. However, in the case of MAP, no specific inhibitors were available for such studies. Instead, a genetically attenuated MAP strain was generated in which MAP expression was placed under the control of an inducible arabinose promoter. Thus, MAP inhibition in whole cells could be mimicked by growth in the absence of arabinose. This genetically attenuated strain was used as a benchmark for MAP inhibition by profiling whole-cell lysates for unprocessed proteins using surface-enhanced laser desorption ionization-time of flight mass spectrometry (MS). Eight proteins between 4 and 14 kDa were confirmed as being unprocessed and containing the initiator methionine by adding back purified MAP to the preparations prior to MS analysis. Upon establishing these unprocessed proteins as biomarkers for MAP inhibition, the assay was used to screen small-molecule chemical inhibitors of purified MAP for whole-cell activity. Fifteen compound classes yielded three classes of compound with whole-cell activity for further optimization by chemical expansion. This report presents the development, validation, and implementation of a whole-cell inhibition assay for MAP.

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In a gene therapy trial with patients suffering from X-linked Severe Combined Immunodeficiency (X1-SCID) led by Fischer and Cavazzana-Calvo (Hacein-Bey-Abina et al., Science, 2003) full restoration of the immune function was observed after retrovirally mediated gamma c transfer. However, three years after treatment, two out of eleven patients developed a T cell lymphoproliferative disorder, which was associated with LMO2 activation as a result of integration of the retroviral vector into the LMO2 locus. LMO2 is required for normal hematopoiesis and is usually only expressed in erythroid cells and immature T-cells as a component of a multifactorial transcription regulation complex consisting of TAL1, LMO2 (mediating protein-protein interaction), GATA1/2, Ldb-1, and E2A. A number of studies with transgenic mice and observations in T-ALL patients suggest that in addition to aberrant LMO2 expression, secondary events, such as mutations in an oncogene like SCL (TAL1) or in tumor suppressor genes are responsible for the onset of malignancy. The goal of this project is to unravel on molecular level specific events which might occur after retroviral mediated gamma c transfer and to determine possible secondary independent events which finally lead to uncontrolled clonal T-cell proliferation. Experiments were initiated with the T-cell clone of one of the patients (patient 5) who developed leukemia-like symptoms after gene therapy. Gene transcription profiling using whole genome gene chips (Affymetrix) revealed, that in addition to LMO2, TAL1 as well as RALDH2 (retinaldehyde dehydrogenase) were among genes which were aberrantly expressed in the patient 5 T-cell clone, whereas in normal T-cell controls none of the three genes were transcribed. By immunoblot analyses and RT-PCR we were able to confirm over expression of LMO2 and TAL1 in the patient lymphoblasts as well as in Jurkat (T-ALL cell line) and K562 (erythroleukemia cell line) cells. In the patient T-cell clone and in the Jurkat cells, RALDH2 was found to be expressed as an N-terminal truncated form on both the mRNA and protein level, suggesting that the molecular mechanism leading to the T-ALL-like lymphoproliferation in patient 5 resembles the findings in T-ALL cell lines described by Ono et al., (Molecular and Cellular Biol., 1998). Ono et al. identified N-terminal truncated (T) RALDH2 as a target gene for a protein complex consisting of LMO2, TAL1, E47 and GATA3 in T-ALL cell lines, in which GATA 3 mediates DNA binding of the complex. Retinoic acid is known to induce cell proliferation and to inhibit activation induced apoptosis of T-cells. We have cloned and overexpressed the T-RALDH variant by transfection and retrovirus transduction to test whether the truncated form of RALDH2 is still capable of converting retinal to retinoic acid and to establish siRNA procedures to examine whether depletion of T-RALDH2 in T-ALL cells changes cellular proliferation.

Three and five years after an otherwise successful gene therapy trial for X1-SCID (gamma c deficiency) three out of 11 patients developed T cell leukemia due to insertional activation of the proto-oncogene LMO2 after retroviral mediated gamma c transfer into autologous CD34+ cells. In one of the patient’s T-cells SCL(TAL1) was expressed in addition to LMO2 because of a SIL-SCL fusion, where the deletion of the SIL gene brings SCL(TAL1) under the control of the constitutively active SIL promoter. LMO2 and SCL(TAL1) are components of a multifactorial transcription regulation complex consisting of SCLTAL1, LMO2, GATA1/2, Ldb-1, and E2A which is essential for normal hematopoiesis; however, LMO2 and SCL(TAL1) are normally down regulated after the DN2 stage of T cell maturation and expression of both proteins after DN2 leads to a block of T cell differentiation which precedes the onset of malignancy as shown in transgeneic mouse models. Experiments with T cell lymphoblasts of the patient, who developed LMO2/SCL(TAL1) associated T-ALL after gene therapy were initiated, in order to determine genes which are deregulated by aberrant expression of LMO2/SCL(TAL1) in mature T-cells leading to the onset of T-ALL. We established a xenotransplant model in NOD/SCID mice with the patient’s leukemic T-cells, which will serve as an in vivo model to examine the mechanisms underlying LMO2/SCL(TAL1) associated T-ALL. By immuno precipitation using LMO2 specific antibodies and nuclear extracts of the patient’s leukemic T-cells, we were able to isolate a protein complex containing LMO2, SCL(TAL) and E47 which resembles the LMO1 associated protein complex described by Ono et al. for the T-ALL cell line Jurkat. Gene transcription profiling using whole genome gene chips (Affymetrix) revealed, that in addition to LMO2 and TAL1, RALDH2 (retinaldehyde dehydrogenase) were among genes which were aberrantly expressed in the patient 5 T-cell clone, whereas in normal T-cell controls none of the three genes are transcribed. Immunoblot analyses and RT-PCR revealed an N-terminal truncation of RALDH2 (T-RALDH2) in the patient’s T-ALL cells. A cellular based activity test using protein extracts of the patient’s T-cells, K562 cells (positive control) and HL-60 cells (negative control) revealed, that the truncated form of RALDH2 is enzymatically active and converts retinaldehyde into retinoic acid which was described to induce cell proliferation and to inhibit activation induced apoptosis of T-cells. To test the role and regulation of RALDH2 in T-ALL we designed RALDH2 and LMO1 specific siRNA for down regulation of RALHD2 and LMO1 in a T-ALL cell culture model (Jurkat cells). We found that down regulation of LMO1 led to a decrease of RALDH2 expression whereas SCL(TAL1) and house keeping genes were not affected. This result confirms the finding of Ono et al. who showed that LMO and SCL(TAL1) activate a truncated form of RALDH2. Both, down regulation of LMO1 or RALDH2 resulted in a decrease of cell viability between 24 and 48 hours post siRNA transfection. We conclude that RALDH2 is a target gene of an LMO1(2)/SCL(TAL1) associated transcription regulating complex in T-ALL and might play a crucial role in the onset of TALL by interfering with proliferation and apoptotic processes.